EP0327797B1 - Method for the preparation of proteins or protein-containing gene products - Google Patents

Abstract

In the prodn. of proteins or protein-contg. gene products by transformation of eucaryotic cells with recombinant DNA contg. the appropriate gene, cultivation of the transformants and isolation of the expressed gene product, the new feature is that cells of a yeast strain deficient in protease A and B are used as host. The host may also be deficient in protease D and carboxypeptidase Y and/or S, e.g. the strain ABYSD-11 (DSM 4322).

Description

The invention relates to a method for manufacturing of proteins or protein products containing genes genetic engineering through transformation of eukaryotic Host cells with the gene for the desired one Protein-containing recombinant DNA molecule, Culturing the cells and isolating the gene product according to known methods.

Clinical-chemical parameters are determined nowadays on a large scale with enzymatic methods. The reagents needed to make them Enzymes are used from various sources of vegetable or animal origin or from microorganisms won.

Microorganisms are becoming increasingly important for the production of enzymes and other proteins, since only these can be made available in virtually any amount by fermentation and thus enable larger amounts of protein to be isolated. As is known, yeasts, such as Saccharomyces cerevisiae, are of particular importance, since in these organisms the homologous expression of proteins is just as possible as the heterologous expression of eukaryotic, for example therapeutically important proteins. In contrast, in E. coli, the most frequently used host organism, many heterologously expressed proteins differ from their natural counterparts expressed in a homologous system and are not biologically active, or are obtained as insoluble inactive protein aggregates, called "refractile bodies". Many eukaryotic proteins that are inactively expressed in E. coli are soluble and actively formed in S. cerevisiae (Biotechnology and Genetic Engineering Reviews 3 (1985) 377-416). Among other things, this may be due to the fact that yeasts have the typical eukaryotic post-translational modification systems, such as "protein folding", protein maturation, glycosylation and acetylation, are capable of secretion and enable the formation of disulfide bridges in polypeptides and proteins. In addition, yeasts are not pathogenic and, in contrast to E. coli, they are free of toxins and pyrogenic cell wall components.

Yeast is one of the oldest cultivated organisms in the world People. It was and still is mainly for alcoholic fermentation (wine, beer, etc.) and as "Baking aid" used in the preparation of pasta. In addition, yeast has industrial importance as Inexpensive raw material source for the isolation of low molecular weight Substances such as NAD, ATP and glutathione, and high molecular substances such as DNA, RNA and especially enzymes, such as Alcohol dehydrogenase, Aldehyde dehydrogenase, acetyl-CoA synthetase, α-glucosidase, Glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase and hexokinase. yeast is easy to cultivate and due to long standing Experience easily fermented on an industrial scale. The yeast, one of the lower eukaryotes single cell counting, has the typical characteristics of a eukaryote, but is still genetic testing and genetic manipulations easily accessible, which makes them particularly as a host organism in the In terms of recombinant DNA technology, i.e. for homologous and heterologous expression of biologically active polypeptides and proteins.

When expressing proteins in yeast is in many However, the amount of protein formed does not fall satisfying. It is often found that after reaching the early inpatient growth phase the specific activity of a desired protein in the course of the stationary Growth phase decreases again, although the educated Biomass of the microorganisms is still increasing. This is et al more host-specific to a proteolytic attack Proteases attributed to the proteins formed.

The object of the present invention was therefore to Process for the production of proteins on genetic engineering Provide ways with which also in the stationary phase of growth proteins formed and can be stably accumulated and thus the yield of the fermentation process can be increased.

This object is achieved by a Process for the production of proteins or proteinaceous Gene products through transformation of eukaryotic Host cells with the gene for the desired one Protein-containing recombinant DNA molecule, cultivation the cells and isolation of the gene product the expression, which is characterized in that one uses a yeast strain as host cells, which is in the stationary breeding phase is deficient in proteases A and B.

The yeast strain is preferably additionally deficient in the Protease D.

In a further preferred embodiment, a yeast strain is used which, in addition to the deficiencies in proteases A, B and optionally D, is deficient in at least one of the carboxypeptidases Y and S. The name of the proteases and carboxypeptidases in this application corresponds to their name in Yeast 1 (1985) 139-154.

By using these protease-deficient yeast strains in the method according to the invention it is possible to Proteins or protein products in yeast to produce in increased yields without extremely long fermentation times or during the subsequent one Isolation according to known methods proteolytic attack, and thus inactivation of proteins.

In a particularly preferred embodiment of the invention the yeast strain is used as host cells ABYSD-11, DSM 4322. This host strain is related to the Proteases A, B, D and the carboxypeptidases Y and S deficient. In addition, he shows auxotrophies in the Adenine, histidine and lysine biosynthesis. auxotrophy means the inability of microorganisms (mostly mutants of bacteria or yeast), certain Growth factors such as Amino acids, from simple To be able to synthesize precursors. In contrast to the corresponding wild type strains grow auxotrophs Mutants are not on so-called minimal media. Instead, they need a full medium that is suitable for the growth necessary components that they don't can synthesize itself contains. microorganisms can for one, but also several growth factors be auxotrophic (E.-L. Winnacker, genes and clones, 1985, Verlag Chemie, Appendix C).

In a further preferred embodiment of the invention crosses the protease-deficient yeast strain another, auxotrophic and / or chemical sensitive Yeast strain, isolated after sporulation by selection on auxotrophy and / or sensitivity from the resulting hybrid strains a yeast strain that at least is deficient in proteases A and B and at least one of the auxotrophies and / or chemical sensitivities of the parent strains and uses this hybrid strain as host cells.

Chemical sensitivity means the inability of a microorganism to grow in a medium that contains certain chemicals, e.g. methotrexate, chloramphenicol and the gentamycin derivative G418. The microorganism can only grow in the medium after transformation of the microorganism with a recombinant DNA which contains a gene which gives the microorganism resistance to these chemicals (dehydrofolate reductase, DHFR; chloramphenicol acetyltransferase, CAT; transposon Tn601-encoded aminoglycoside phosphotransferase etc.) , The crossing and sporulation can be carried out, for example, analogously to Sherman et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1984. In a particularly preferred embodiment of the invention, the yeast strain ABYSD-11, DSM 4322, ( a pra1 , prb1 , prc1 , prd1 , cps1 , ade lys his7 ) is crossed with either the yeast strain TCY2824-1A ( α mal1S-Δ ura3- 52 his4 ), DSM 4317, or DBY 746, DSM 4316, ( α his3-Δ1 leu2-3 leu2-112 ura3-52 trp1-289a ), which contains a defective maltase structural gene and auxotrophies in the uracil and histidine or auxotrophies in the Show histidine, leucine, uracil and tryptophan biosynthetic pathway. Hybrid strains are then preferably isolated which are deficient in proteases A, B and, if appropriate, D, and in carboxypeptidases Y and S, and additionally at least one or more auxotrophies, such as, for example, the uracil, lysine and maltase utilization auxotrophs, or the leucine and Tryptophan, or finally the leucine, uracil and histidine auxotrophies of the parent strains.

In a further preferred embodiment of the invention therefore contains the recombinant DNA molecule in addition to the gene for the desired protein or protein-containing gene product one or more genes that the auxotrophies and / or chemical sensitivities of the Complement the host strain.

The preferred embodiment of the invention a simple distinction between transformed and not transformed host cells. The Presence and expression of genes that one or several of the auxotrophies and / or chemical sensitivities complement of the host strain, enables namely transformed cells, also towards media grow, for example, an amino acid contain that the host strain itself does not synthesize can, however, the gene on the recombinant DNA molecule is present. Non-transformed host strains, which is the recombinant DNA molecule and the auxotrophy-complementing gene contained on it can not have, however, in such a Medium does not grow. This can be done in a simple manner selection for transformed host cells be made, which also creates the risk of Loss of the recombinant DNA which is the gene for the contains desired protein during fermentation is avoided since no growth advantage of non-transformed Cells.

Alternatively, it is also possible to use auxotrophy or / and Chemical sensitivity of a host strain by introduction another additional recombinant DNA molecule that contains one or more genes that the auxotrophies or chemical sensitivities of the Complement host cells to overcome. It can however, the easy way of selection host cells containing the gene for the desired product not be noticed.

All recombinant come for the recombinant DNA molecule DNA molecules in question with which yeast cells can be transformed and used for expression are capable of a foreign gene. Here comes not only that extrachromosomal transcription, for example one Plasmids in question but it is also possible the gene for the desired protein via an integration vector or an integrating DNA fragment that one complete expression cassette (promoter, Terminator, regulator, transcription enhancer, etc.) included to infiltrate the yeast genome and together to express with the yeast's own proteins. requirement for this is the presence of homologies on the recombinant DNA molecule and chromosomal Yeast sequences. Such a homologous region can be used integrating DNA fragment according to known Methods are introduced into the yeast chromosome.

In a further preferred embodiment of the invention, the recombinant DNA molecule is therefore either a plasmid or else an integration vector or an integrating DNA fragment. Again, yeast plasmids are particularly preferred as plasmids, which occur in the cell in high number of copies. Such yeast plasmids are, for example, hybrid yeast / E. coli vectors ("shuttle vectors"), which are referred to as YRp, YEp, YIp and YCp. In turn, only the YEp and YRp plasmids occur in high number of copies in the cell. This is because, due to the presence of sequences which enable the plasmids to replicate independently, they are independent of the replication of the yeast chromosome and are usually present in quantities of 5 to 40 copies per cell. The YIp plasmids can only be expressed by integration into the yeast genome. They are therefore exemplary. for integration vectors. YIp plasmids have a ten times lower transformation rate, but a significantly greater stability compared to YRp and YEp plasmids. YRp and YEp plasmids can be lost in cell growth without selection pressure (Nature 305 (1983) 391-397). Such a selection pressure exists, however, in the preferred embodiment of the invention, in which the fermentation of auxotrophic or chemical-sensitive host strains is carried out in minimal media, or media which contain a certain chemical for which the host strain is sensitive, and additionally the recombinant DNA or has multiple genes that complement auxotrophy or sensitivity.

The present invention enables expression of homologous or heterologous proteins or protein-containing Gene products in high yield and proteolytic not attacked form, including the selection on transformed cells, and thereby also one Increasing the yield of the desired gene product, in can be done easily. Also at Unlocking the cell mass and the further procedure to obtain the gene product according to known Methods occur due to the protease deficiency of the Host cells no proteolytic attack on the formed Product.

In a preferred embodiment of the invention the protein alpha-glucosidase PI is produced (see also example 4).

The following examples further illustrate the invention.

example 1

For the production of the Saccharomyces strains ABYSMAL81 and ABYSDMAL81, the haploid Saccharomyces cerevisiae strain ABYSD-11 ( a pra1 prb1 prc1 prd1 , cps1 , ade lys his7 ), DSM 4322, the proteases A, B and D and the carboxypeptidases Y and S were used is deficient, and additionally has auxotrophy in adenine, histidine and lysine biosynthesis, with the Saccharomyces carlsbergensis strain, TCY2824-1A, ( α mal1S-Δ ura3-52 his4 ), DSM 4317, crossed by a defective α-glucosidase structural gene and is characterized by auxotrophy in uracil and histidine biosynthesis.

A sporulation was then carried out and the yeast segregants formed were checked for their auxotrophies by plating on various selection media and for their protease deficits by determining the protease activities in cell lysates. The strains ABYSDMAL81 ( ura3-52 times1S-Δ lys pra1 prb1 prc1 prd1 cps1 ) and ABYSMAL81 (ura3-52 times1S-Δ lys pra1 prb1 prc1 cps1 ) were identified by:

Evidence of protease deficits

The segregants were placed in 5 ml of YEPD medium (1% yeast extract, 2% peptone, 2% glucose), the cells in the late logarithmic to early stationary growth phase harvested, washed twice with water and with glass beads by homogenizing on a whirl mix open-minded (MGG 145 (1976) 327-333). The cells were extracted with 1 ml of 20 mmol / l Tris (HCl), pH 7.0 and the supernatant after centrifugation as a crude extract further processed. To activate the proteases the crude extract titrated to pH 5.0 and at 24 hours Incubated at 25 ° C.

Detection of protease A deficiency (pra1)

No hydrolysis by cell extracts of 1.2% acid-denatured Hemoglobin, pH 3.0 (Eur. J. Biochem. 42 (1974) 621-626).

0.5 ml 0.1 mol / l lactate buffer, pH 3.0 with 1.2% acid-denatured Hemoglobin was added with 0.1 ml of cell lysate Incubated at 25 ° C. After 30 minutes the reaction started with 0.5 ml of 10% trichloroacetic acid stopped and after Centrifugation of the trichloroacetic acid soluble products either by absorption measurement at 280 nm or by a modified Folin determination according to McDonald and Chen (Anal. Biochem. 10 (1965) 175-177).

To calculate the specific activity, a Protein determination according to Zamenhof carried out (Methods Enzymol. 3 (1957) 702).

Protease A deficiency exists when the specific hydrolytic activity of cell lysates towards acid-denatured hemoglobin compared to less than 5% is degraded with a wild type strain.

Detection of protease B deficiency (prb1)

No hydrolysis by cell extracts of 2.4% Azocoll pH 7 (Eur. J. Biochem. 42 (1974) 621-626).

0.5 ml of a 2.4% Azocoll suspension in 0.1 mol / l Phosphate buffer, pH 7.0 was added with 0.1 ml of cell lysate Shake incubated at 25 ° C. To activate the Protease B became the crude extract before the activity determination with sodium dodecyl sulfate (final concentration 0.25%) added.

After 30 minutes the reaction was stopped by adding 0.5 ml of 10% trichloroacetic acid stopped and after Centrifugation of the absorbance in the supernatant at 550 nm certainly.

Protease B deficiency is present when the specific hydrolytic activity of cell lysates towards Azocoll to less than 5% compared to a wild type strain is reduced.

Detection of protease D deficiency (prd1)

No hydrolysis by cell extracts of 0.5 mmol / l Bz-Pro-Phe-Arg-NA (benzoyl-L-prolyl-L-phenylalanyl-L-arginyl-p-nitroanilide) in 50 mmol / l Tris-maleate buffer, pH 7.0 in the presence of aminopeptidase M (J. Biol. Chem. 260 (1985) 4585-4590)

0.03 ml of 0.5 mol / l Tris-maleate buffer, pH 7.0 were added 0.015 ml 10 mmol / l Bz-Pro-Phe-Arg-NA (dissolved in dimethyl sulfoxide), 10 µl (40 µg, 240 mU) aminopeptidase M, 0.145 ml of water and 0.1 ml of crude extract mixed and the Change in extinction at 405 nm against a reagent blank certainly.

A protease D deficiency exists when the specific hydrolytic activity against Bz-Pro-Phe-Arg-NA to less than 10% compared to a wild type strain is reduced.

Detection of carboxypeptidase Y deficiency (prc1)

No hydrolysis by cell extracts of 0.5 mmol / l Benzoyl-L-tyrosine-4-nitroanilide, pH 7 (Agr. Biol. Chem. 35 (1971) 658-666).

0.1 ml activated with deoxycholate (final concentration 0.5%) crude extract were with 1 ml 0.1 mol / l phosphate buffer pH 7.0 and 0.2 ml of 3 mmol / l benzoyl-L-tyrosine-4-nitroanilide (dissolved in dimethylformamide) incubated at 25 ° C. To For 10 minutes, the reaction was with 1 ml of 1 mmol / l mercury chloride stopped and the released p-nitroaniline determined at 410 nm.

Carboxypeptidase Y deficiency is present when. the specific hydrolytic activity towards benzoyl-L-tyrosine-4-nitroanilide to less than 5% compared to a wild type strain is degraded.

Detection of carboxypeptidase S deficiency (cps1)

No hydrolysis by cell extracts from Cbz-Gly-Leu (Benzyloxycarbonyl-glycyl-L-leucine) at pH 7.4 with subsequent analysis of the released leucine in an L-amino acid oxidase-peroxidase test (Eur. J. Biochem. 73 (1977) 553-556).

0.5 ml test solution (0.25 mg / ml L-amino acid oxidase, 0.4 mg / ml horseradish peroxidase and 0.5 mmol / l MnCl ₂ ), 0.4 ml 27.5 mmol / l Cbz-Gly- Leu solution (dissolved in 0.2 mol / 1 phosphate buffer, pH 7.0), 0.05 ml o-dianisidine dihydrochloride (2 mg / ml, dissolved in H ₂ O), 0.05 ml 22 mmol / l phenylmethylsulfonyl fluoride and 0 , 1 ml of dialysed cell lysate (dialysis: 0.1 M imidazole chloride, pH 5.3; 24 hours; 25 ° C.) and the change in extinction at 405 nm was determined.

Carboxypeptidase Y deficiency is when the specific hydrolytic activity towards Cbz-Gly-Leu to less than 5% compared to a wild type strain is reduced.

Based on the described evidence, it was determined that the ABYSDMAL81 strain deficient in proteases A, B, D and the carboxypeptidases Y and S and the strain ABYSMAL81 deficient on proteases A, B and Carboxypeptidases Y and S.

Evidence of maltose utilization auxotrophy

No growth on complete synthetic medium I with 0.67% yeast nitrogen base (YNB, salt-vitamin mixture, Difco), 0.5% casamino acids (CAA, protein hydrolyzate, Difco), 2% maltose (only C source), 20 mg / l uracil and 30 mg / l adenine.

Evidence of uracil auxotrophy

No growth on synthetic complete medium II with 0.67% YNB, 0.5% CAA, 2% glucose (sole C source) and 30 mg / l adenine.

Detection of lysine auxotrophy

No growth on synthetic complete medium II with Uracil (20 mg / l) but without lysine (instead of 0.5% CAA an amino acid mixture without lysine was used).

Detection of histidine and adenine prototrophy

Growth on synthetic complete medium II with uracil (20 mg / l) but without adenine and without histidine (instead of 0.5% CAA became an amino acid mixture without histidine used).

Examples 2 and 3

For the production of the saccharomyces strains ABYSD91 ( leu2-3.2-112 trp1-289a pra1 prb1 prd1 prc1 cps1 ), ABYSD106 ( ura3-52 leu2-3.2-112 his pra1 prb1 prd1 prc1 cps1 ), ABYS91 ( leu2-3.2 -112 trp1-289a pra1 prb1 prc1 cps1 ) and ABYS106 ( ura3-52 leu2-3.2-112 his pra1 prb1 prc1 cps1 ) was described as in Example 1, the Saccharomyces cerevisiae strain ABYSD-11 with the Saccharomyces carlsbergensis strain DBY746, DSM 4316, crossed. After sporulation, the yeast plants were checked for their protease deficits and auxotrophies.

For ABYS91 and ABYSD91: Evidence of protease deficits See example 1 Detection of leucine and tryptophan auxotrophy

No growth on synthetic complete medium II with Uracil, but without leucine or tryptophan (instead of 0.5% CAA was an amino acid mixture without leucine or Tryptophan used).

Detection of uracil, adenine, histidine and lysine prototrophy

Growth on synthetic complete medium II without Adenine, uracil, histidine and lysine (instead of 0.5% CAA became an amino acid mixture without histidine and Lysine used).

For ABYS106 and ABYSD106 Evidence of protease deficits See example 1 Evidence of uracil auxotrophy

No growth on synthetic complete medium II.

Detection of leucine and histidine auxotrophy

No growth on synthetic complete medium II with Uracil but without leucine or histidine (instead of 0.5% CAA was an amino acid mixture without leucine or Histidine used).

Detection of adenine, lysine and tryptophan prototrophy

Growth on synthetic complete medium II with uracil, but without adenine, lysine and tryptophan (instead of 0.5% CAA became an amino acid mixture without lysine and Tryptophan used).

Example 4 Expression of α-glucosidase PI

The Saccharomyces strain ABYSMAL81 (Example 1) was transformed with the plasmid YEp / 5C6b3 (Nature 275 (1978) 104-109).

To prepare this plasmid, the vector YRp / GLUPI, DSM 4173P, was digested with the restriction endonucleases SspI and HindIII, the approximately 3.0 kBp long SspI / HindIII fragment was isolated and into the isolated PvuII / -SpHI vector fragment from YEp 24 (Gene 8 (1979), 17-24; Cold Spring Harbor, Symp. Quant. Biol. 43 (1979) 77-90; Gene 29 (1984) 113-124; Nature 286 (1980) 860-865) - after replenishment the overhanging 5 'end of the HindIII and degradation of the overhanging 3' end of the SphI restriction site with Klenow polymerase - ligated. The α-glucosidase PI expression cassette is integrated in the resulting plasmid YEp / S4 in the same orientation as the β-lactamase gene. An approximately 3.1 kbp long BamHI fragment containing the MAL2-8 ^C p gene was then ligated into the BamHI restriction site of YEp / S4. For this, the plasmid pRM2, DSM 4314P, was digested with the restriction endonuclease SalI, the overhanging 5 'ends were filled in with Klenow polymerase, provided with BamHI linkers (d (CGGGATCCCG)), cleaved with BamHI and the 3.1 kBp long MAL2 BamHI fragment containing -8 ^C p gene isolated. The resulting vector construction was designated YEp / 5C6b3.

The transformed strain was in YEP medium (1% Yeast extract, 2% peptone) with 4% maltose and up to the late logarithmic or stationary phase bred. The biomass was then harvested and washed with 10 mmol / l phosphate buffer, pH 6.8. The Cells from 5 ml YEP medium (approx. 0.1 to 0.2 g yeast, Wet weight) were obtained by homogenizing with a Whirlmix digested (MGG 145 (1976) 327-333).

The specific α-glucosidase activity was determined using the hydrolysis of p-nitrophenyl-α-D-glucopyranoside (MGG 151 (1977) 95-103) and the protein determination according to Zamenhof (Methods Enzymol. 3 (1957) 702).

That was in the crude extract obtained in this way Enzyme stable for 10 days at 4 ° C. Also in SDS gel electrophoresis there was no change over this period of the band pattern found. This shows that too the other enzymes and in this supernatant Proteins in this yeast strain are stable and not are noticeably proteolytically degraded.

Table I compares the enzymatic stability of the baker's yeast α-glucosidase (obtained from Deutsche Hefewerke Nürnberg, DHW) with the stability of recombinantly expressed α-glucosidase in protease-deficient α-glucosidase transformants. In baker's yeast, the specific α-glucosidase activity reaches a maximum in the late logarithmic to early stationary growth phase. As the fermentation progresses, the specific α-glucosidase activity drops markedly (Table 1). In contrast to this, surprisingly, even after reaching the stationary growth phase, the α-glucosidase is stably accumulated in transformed protease-deficient malO strains, which considerably simplifies the fermentation and processing of the biomass.

Fermentation media: baker's yeast: 1% yeast extract, 2% Peptone, 2% maltose.

ABYSMAL81: Complete synthetic medium II.

Example 5

Heterologous expression of a fusion protein, consisting of from the N-terminal part of the α-glucosidase and HIV1 antigens, in protease deficient yeast strains.

In the α-glucosidase PI expression vector YEp / 5C6b3 (Example 4) the 1.4 KBp BglII fragment, which codes for approximately 80% of the α-glucosidase PI against a approx. 300 bp long DNA fragment, which is used for part of the gp41 membrane protein of the HIV1 retrovirus coded, exchanged. For this purpose, an approximately 300 bp long RsaI / HindIII fragment (Sequence see sequence of WMJ-1 from item 1638 to Pos. 1943 from Fig. 1 by Cell 45 (1986) 637-648) in the E. coli vector digested with HincII and HindIII pUC18 (M13mp18 and pUC19, sequence in Gene 33 (1985) 103-119) subcloned (construction: pUC18HRH.300). Out pUC18HRH.300 became the approximately 320 bp BamH1 / HindIII fragment isolated and into the approx. 5.2 KBp long pUR278 BamH1 / HindIII vector fragment ligated (sequence in EMBO 2 (1983) 1791-1794) (Construction: pUR278HRH.300). The Plasmid pUR278HRH.300 was digested with HindIII overhanging 5 'ends filled with "Klenow polymerase" and provided with BamHI linkers (d (GGGATCCC)). Then it was cleaved with BamHI, the approx. 300 bp long BamHI fragment isolated and into the approx. 11 KBp long YEp / 5C6b3 BglII vector fragment ligated. at correct orientation of the gp41 membrane polypeptide DNA 'a fusion protein is created, consisting of the N-terminus of α-glucosidase (50 amino acids), 4 construction-related Amino acids at the fusion site, 101 amino acids of the gp41 membrane protein and 3 design-related Amino acids at the C-terminus with a molecular weight of approx. 18500 D. The desired construction was over the expressed fusion protein in the protease deficient Yeast expression strain ABYSMAL81 after transformation and cultivation (see below) followed by SDS gel electrophoresis and Western blot based on immunoreactivity isolated with human HIV1 sera. The fusion protein was expressed to about 5% of the total protein and was as the dominant band in SDS polyacrylamide gels Coomassie staining visible.

To express the α-Gluc.PI-gp41 fusion protein, the transformants were grown on selective medium (0.67% YNB, 0.5% CAA, 30 mg / l adenine) with 2% glucose and 2% maltose. After an induction phase of 10 to 20 hours (after glucose consumption), the cells were harvested.

Claims (10)

1. Method for producing proteins or gene products containing protein by transforming eukaryotic host cells with a recombinant DNA molecule containing the gene for the desired protein, culturing the cells and isolating the gene product after expression, characterized in that a yeast strain which is deficient in proteases A and B in the stationary growth phase is used as the host cells.
2. Method as claimed in claim 1, characterized in that the yeast strain is additionally deficient in protease D.
3. Method as claimed in claim 1 or 2, characterized in that the yeast strain is additionally deficient in carboxypeptidases Y or/and S.
4. Method as claimed in claim 3, characterized in that the yeast strain ABYSD-11, DSM 4322 is used.
5. Method as claimed in one of the claims 1 to 4, characterized in that the protease-deficient yeast strain is crossed with another auxotrophic or/and chemical-sensitive yeast strain, after sporulation and selection of the resulting hybrid strains by means of the auxotrophy or/and chemical sensitivity, a yeast strain is isolated which is at least deficient in proteases A and B and has at least one of the auxotrophies or/and chemical sensitivities of the parent strains and this hybrid strain is used as the host cells.
6. Method as claimed in claim 5, characterized in that the yeast strain ABYSD-11, DSM 4322 is crossed with the strain TCY2824-1A, DSM 4317 or DBY746, DSM 4316, a strain which is deficient in proteases A, B and the carboxypeptidases Y and S and optionally protease D and has the uracil, lysine and maltose utilization auxotrophies, the leucine and tryptophan or leucine, uracil and histidine auxotrophies of the parent strains is isolated and used as host cells.
7. Method as claimed in one of the claims 5 or 6, characterized in that a recombinant DNA molecule is used which additionally contains one or more genes which complement the auxotrophies or/and chemical sensitivities of the host strain.
8. Method as claimed in one of the claims 1 to 7, characterized in that a plasmid, an integration vector or an integrating DNA fragment is used as the recombinant DNA molecule.
9. Method as claimed in claim 8, characterized in that the recombinant DNA molecule is a yeast plasmid which occurs in a high copy number in the cell.
10. Yeast strain which is deficient in proteases A and B in the stationary growth phase.